PROJECT SUMMARY The Blenner Research Group at Clemson University is broadly focused on using genetic engineering and synthetic biology to improve microbial systems for applications in human health, sustainability, and national defense. Our research is aimed at developing the capabilities to accurately and precisely control the properties of oleaginous yeast ? Yarrowia lipolytica. This yeast has naturally evolved the capability to produce a significant amount of lipid. As such, they are useful for producing biochemicals derived from lipids, such as omega-3 fatty acids and fatty alcohols. As oleaginous yeast are considered non-conventional and in many cases non-model systems, there were few genetic engineering tools available to manipulate these systems. The Blenner Research Group has invested significant time and energy building finely-tuned promoters, inducible promoters, temporal controlled promoters, and a suite of CRISPR-Cas9 technologies for rapid genome editing. Significant progress in lipid and oleochemical production was due to Y. lipolytica's innate high flux capacity through intermediates and cofactors needed for lipid biosynthesis. Our broad goal over the next five years are to increase the diversity of products that are made in oleaginous yeast. There are over 200,000 known natural products structures and natural products make up the majority of therapeutic compounds. Many bioactive natural products are derived from plants; however, isolation from plants is uncertain, can be extremely costly, and in some cases, agriculture cannot provide enough material. Several classes of plant natural products, including terpenoids, flavonoids, and alkaloids, are biosynthesized using fundamental building blocks from high flux pathways in Y. lipolytica. As such, we hypothesize oleaginous yeast are ideally suited for high titer natural product biosynthesis. This proposal focuses on addressing a common problem across all natural product heterologous pathways ? difficulty expressing foreign proteins. We seek to identify known and novel responses to common mode of heterologous protein expression failure. Once specific genes associated with each modes of failure are identified, these can serve as indicators to identify a new gene's mode of failure. These can also be used to help identify solutions to improve expression based on the mode of failure. The resulting improvements can be stacked into platform strains and used to more easily screen natural product pathway libraries. We will focus on flavonoids due to their diverse chemistry and known therapeutic effects; however, we expect these platform strains to be useful for making other natural products as well.